4.5 ASSISTED REPRODUCTION FOR THE INFERTILE CROSS

4.5.1 Artificial insemination

Although artificial insemination is a critical tool for reproductive biologists
working with other species (including humans), it is not often used by mouse
geneticists. Its major use in other species is to initiate a successful pregnancy when,
for any of a number of reasons, the male cannot or should not, be directly involved
in the process of mating. Artificial insemination has been a boon to the cattle
industry because the semen from one good bull can be shipped around the world to
impregnate unlimited numbers of females. Male mice are somewhat smaller than
bulls and, as a consequence, the whole animal can be shipped for a cost that is likely
to be the same (or less) than one would pay for frozen semen alone. Furthermore,
obtaining semen from a mouse is a "one-shot" deal. Since assisted masturbation of
the male mouse is not practical, sperm must be recovered from the epididymis after
the animal has been sacrificed.

There are some special cases where artificial insemination can be used as an
experimental tool for the study of the mouse. One example is in those cases where,
for behavioral reasons, males of a particular strain refuse to mate with selected
females of another strain. This scenario is most likely to occur when the males and
females are members of different Mus species.
West and colleagues (1977) used
artificial insemination to overcome this problem in order to determine the viability
of various hybrid embryos formed between distantly related members of the Mus
genus.

Another use of artificial insemination is in those cases where one wants to alter
the composition of the sperm pool. For example,
Olds-Clarke and Peitz (1985)
were able to analyze the relative fertilizing potentials associated with sperm
obtained from two different males by mixing equal numbers together before
insemination. Finally, there will always be the case where a one-of-a-kind male  such
as a first generation transgenic or another new mutant  refuses to participate
in the mating process. As a last resort, one can recover epididymal sperm from such
an animal for a single chance at achieving a pregnancy. Detailed protocols for sperm
recovery and artificial insemination have been described elsewhere
(Rugh, 1968;
West et al., 1977;
Olds-Clarke and Peitz, 1985).

When a choice is possible, females to be inseminated should not be inbred; F1
hybrids and random-bred animals will always have higher levels of fertility. A
successful fertilization can only occur when the inseminated female is in the late
proestrus/early estrus stage of the estrus cycle. Appropriately staged females can be
obtained either by visual inspection of naturally cycling animals (as described
earlier in this chapter)
or through superovulation (see
Section 6.2). The implantation of
fertilized embryos will occur only in females that have been stimulated into a state
of pseudopregnancy
(Section 6.2.3). If the investigator intends to use a sterile stud
male for this purpose, the mating should be performed after the insemination
(within 0.5-2 hours) so that the vaginal plug does not interfere with the protocol
(Olds-Clarke and Peitz, 1985).
If pseudopregnancy is to be induced manually, it
should be accomplished in the fully alert female prior to the insemination protocol (see
Section 6.2.3 for details).

4.5.2 Transplantation of ovaries

In a small number of instances, females that express certain mutations may be
fertile in the sense that they are able to produce functional oocytes but infertile in
the sense that they are physically unable to bring offspring to term. Such females
may not be able to mate, they may not be fit enough to allow gestation to proceed
properly or they may be unable to birth live offspring.

4.5.3 In vitro fertilization

A third method of assisted reproduction entails fertilization outside the female
reproductive tract. There are two general types of circumstances when in vitro
fertilization becomes useful: when the female partner of a cross is unable to carry
litters to term for one reason or another; and when an investigator wants to
establish the timing of fertilization to a more precise degree and/or wants to
synchronize the development of a batch of embryos for later recovery and analysis.
A detailed discussion of this procedure and all other aspects of embryo
manipulation are provided in the manual by
Hogan and her colleagues (1994).